Bioavailability and bioactivity of small molecules, peptides, proteins or nucleic acids can be altered by glycosylation. Glycosylation is the transfer of an activated sugar moiety from a donor to an acceptor molecule and is catalysed by glycosyltransferases. Glycosylation of proteins occurs either co- or post-translationally by which a sugar moiety is attached to a nitrogen of asparagine or arginine side-chains forming N-linked glycans, or to the hydroxy oxygen of serine, threonine, tyrosine, lysine, or proline side-chains forming O-linked glycans, or less common forming C-linked glycans where the sugar is added to carbon on a tryptophan side-chain.
N-linked glycosylation is the most common post-translational modification and is carried out in the endoplasmic reticulum of eukaryotic cells. N-linked glycosylation can be of two main types; high mannose oligosaccharides which have two N-acetylglucosamines and complex oligosaccharides which include other types of sugar groups. The peptide motif contained in glycosylated polypeptides is Asn-X-Ser or Asn-X-Thr where X is any amino acid except proline. This is catalysed by the enzyme oligosaccharyltransferase [OT]; see Yan & Lennarz J. Bioi. Chem., Vol. 280 (5), 3121-3124 (2005). OT catalyses the transfer of an oligosaccharyl moiety (Glc3Man9GlcNAc2) from the dolichol-linked pyrophosphate donor to the side chain of an Asn. A pentasaccharide core is common to all N-linked oligosaccharides and serves as the foundation for a wide variety of N-linked oligosaccharides. O-linked glycosylation is less common. Serine or threonine residues are linked via their side chain oxygen to sugars by a glycosidic bond. Usually N-acetylglucosamine is attached in this way to intracellular proteins.
Most bacterial glycoproteins are associated with the bacterial cell surface or are secreted, suggesting a role for glycoproteins in the interaction with the host's immune system. Studies on the gram negative pathogenic bacterium Campylobacter jejuni identified a gene cluster involved in the synthesis of lipo-oligosaccharides and N-linked glycoproteins. The protein glycosylation locus, a cluster of 12 genes comprising pglA-pglG, is involved in the glycosylation of over 30 glycoproteins. Interestingly, these genes can be used to modify lipopolysaccharide structures in Escherichia coli providing a genetic tool to express heterologous recombinant glycoproteins. Studies performed with C. jejuni carrying mutations in the pglB and pglE genes showed impaired colonisation abilities in mice.
Polysaccharide antigens interact directly with B cells and induce antibody synthesis in the absence of T-cells. This T-cell independent immune response is limited as antibody production is low and is not normally boosted by re-immunisation. The antibody isotypes are dominated by IgM and IgG2, which are short lived and are generally of low affinity for a specific antigen. The ability to enhance the immunogenicity of polysaccharide antigens can be achieved by conjugation of the polysaccharide to a protein carrier. Glycoconjugate vaccines for Streptococcus pneumoniae, Neisseria meningitidis and Haemophilus influenzae are currently licensed for human use and are produced by linking the capsule (or other bacterial glycan-based structure such as lipooligosaccharide) from these bacteria to a protein toxoid. Whilst these vaccines provide a good level of immunity they are expensive and difficult to produce, requiring the purification of the glycan from the pathogenic organisms and chemical linkage to the carrier protein. The use of organic systems represents a more rapid and economical method for the production of glycoconjugates.
The production of glyconjugates in an E. coli expression system requires the co-expression of three genes [“tri-plasmid”]: an acceptor protein, a polysaccharide biosynthetic locus and, for the coupling reaction, an oligosaccharyltransferase enzyme. Optimisation of co-expression in just one host is a lengthy process as it requires a tailored choice of the plasmids and optimised growth and expression conditions which are dependent on several factors. Additionally, the yield of glycoconjugates using such expression systems are often very low, which makes it commercial not viable.
The tri-plasmid system has a number of disadvantages. Compatible origins of plasmid replication need to be combined, a process that can take several months and restricts the choice of available plasmids. The use of three plasmids represents a considerable metabolic burden on the host bacterial strain and often the contents of one of the plasmids fail to express. Three different antibiotic selection markers for each of the plasmids need to be combined. This represents another burden on the host strain and clashes often result in sub-cloning of antibiotic resistance genes to find combinations that match. Additionally, some antibiotics resistance combinations are legally forbidden in some strains. Some glycosyltransferases [e.g. PgIB enzyme] responsible for linking the glycan to the protein has multiple transmembrane domains and is toxic to bacterial cells. Once expressed it has been found to reduce the growth of the host bacterial strain considerably. This significantly inhibits glycoconjugate yield. Heterologous expression of the polysaccharide coding genes and the resulting enzymes that are generated can result in high levels of toxicity to the host. Therefore, with some glycans it is impossible to find a tri-plasmid combination that allows for glycoconjugate production.
An example of the tri-plasmid system is disclosed in WO2009/104074 which describes the optimised production of conjugates comprising a protein carrier linked to an antigenic polysaccharide in an E. coli tri-plasmid expression system. The yield using this system is increased significantly by growing cells carrying three genes on two plasmids in a bioreactor under carefully monitored growth condition.
The present disclosure relates to a method providing the stable integration of genes into a bacterial genome for the production of glycoconjugates controlled by constitutive and regulated promoter elements which results in high yields of glycoconjugate. The application discloses also a method for the genetic manipulation of transposons facilitating easy and efficient preparation of the transposable element which allows integration of genes into the genome in one single step and additionally comprising also a tool which permits easy removal of the resistance marker once the gene of interest has integrated into the host genome.